Heart disease is the primary cause of death in the United States, and most heart disease can be attributed to coronary artery atherosclerosis. Low HDL-cholesterol is an independent risk factor for coronary artery disease and myocardial infarction. One protective role of HDL is as a mediator of reverse cholesterol transport. Apolipoprotein A-l (apoAl) is the major protein constituent of HDL. ABCA1 is required for apoAl lipidation and the genesis of HDL, as demonstrated by the absence of apoAl lipidation in Tangier disease subjects who have defective ABCA1 and enhanced cardiovascular disease risk. The objectives of this proposal are to understand the mechanism by which ABCA1 mediates the binding of and lipid efflux to apoAl, and apoAl structural features that enable it to act as a lipid acceptor. Achieving the aims of this proposal will allow us to have a more detailed understanding of the process of lipid efflux and HDL biogenesis, and may have an impact on the emerging field of therapeutics designed to raise HDL levels for the treatment and prevention of coronary artery disease. The first aim is to determine the changes in the plasma membrane mediated by ABCA1 that allow recognition by apoAl. This involves lipid, protein, and biophysical studies of the plasma membrane, and liposome studies. The second aim is to understand the role of endocytosis in lipid efflux and the mechanism of assembly of the nascent lipoprotein. This involves inhibition of endocytosis by genetic methods, time course studies of apoAl uptake and release, testing a novel model for lipid assembly onto apoAl, and identification of apoAl binding sites on ABCA1. The third aim is to determine structural features of apoAl and model peptides that are required for them to function as acceptors of cellular lipids. This involves studying the role of helix unfolding and the role of the positivelycharged residues in lipid binding and acceptor activity. In addition, this study will include the identification of the apoAl oxidative modification by the enzyme myeloperoxidase that diminishes its lipid acceptor activity.